Mycophenolic acid has the chemical name 6-[4-Hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl]-4-methyl-hex-4-enoic acid, 6-[1,3-Dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-isobenzofuran-5-yl]-4-methyl-hex-4-enoic acid, molecular formula of C17H20O6, molecular weight of 320.35, CAS Registry number of 24280-93-1 and a structure of:
Mycophenolic acid (MPA), isolated by Gosio in 1893, is the first well-characterized antibiotic (Bentley 2001). It is produced by several species of Penicillium, including P. brevi-compactum, P. scabrum, P. nagem, P. roqueforti, P. patris-mei, and P. viridicatum (Clutterbuck et al. 1932, Jens and Filtenborg 1983).
MPA, in addition to its antibiotic activity (Abraham 1945), also has antifungal (Gilliver 1946), antiviral (Ando et al. 1968), and antitumor properties (Noto et al. 1969), and has been used clinically in the treatment of psoriasis (Johnson 1972). More recently, it has been recognized as a powerful immunosuppressant (Bentley 2000).
At least one reason for its pharmacological properties is the fact that in several biological systems it interferes with guanine biosynthesis at the level of inosine monophosphate dehydrogenase (IMPD). It has, therefore, a pronounced inhibitory effect on nucleic acid synthesis (Franklin and Cook 1969). The inhibition of IMPD is also the basis of its lymphocyte-specific immunosuppressive effect. Since lymphocytes primarily depend on de novo guanine biosynthesis, the reduction of this pathway results in suppression of T and B lymphocyte proliferation.
MPA was withdrawn due to its high incidence of side effects (primarily infections such as herpes zoster and gastrointestinal side effects such as stomach discomfort). The 2-morpholinoethyl ester derivative, mycophenolate mofetil (CellCept®) does not have these drawbacks, and has a better bioavailability than mycophenolic acid. Mycophenolate mofetil was recently approved (in the United States in 1995 and in Europe in 1996) for prophylaxis of organ rejection in patients receiving allogeneic renal transplants (Shaw and Nowak 1995, Sollinger 1995). After oral administration the ester form rapidly hydrolyzes to free acid. MPA is then converted mainly to an inactive glucuronide metabolite, which is eliminated by urinary excretion (Bentley 2001, Wiwattanawongsa et al. 2001).
Chemically, mycophenolate mofetil (abbreviated as MMF) is 2-(4-morpholinyl)ethyl (E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-isobenzofuran-5-yl)-4-methyl-4-hexenoate, and its first synthesis was disclosed in U.S. Pat. No. 4,753,935.

Another patent, U.S. Pat. No. 5,543,408 discloses the anhydrous crystalline salt form, monohydrate salt form, and amorphous salt form of mycophenolate mofetil. These forms are characterized by their melting points and/or Differential Scanning Calorimetric (DSC) results and/or powder X-ray diffraction pattern.
The product mixture of a reaction rarely is a single compound pure enough to comply with pharmaceutical standards. Side products and byproducts of the reaction and adjunct reagents used in the reaction will, in most cases, be present. At certain stages during processing of the mycophenolate mofetil contained in the product mixture into an active pharmaceutical ingredient (“API”), the mycophenolate mofetil must be analyzed for purity, typically by HPLC or GC analysis, to determine if it is suitable for continued processing or ultimately for use in a pharmaceutical product. The mycophenolate mofetil does not need to be absolutely pure. Absolute purity is a theoretical ideal that is unattainable. Rather, there are purity standards intended to ensure that an API is not made less safe for clinical use because of the presence of impurities.
The U.S. Food and Drug Administration's Center for Drug Evaluation and Research (CDER) has promulgated guidelines recommending that drug applicants identify organic impurities of 0.1% or greater in the active ingredient. “Guideline on Impurities in New Drug Substances,” 61 Fed. Reg. 371 (1996); “Guidance for Industry ANDAs: Impurities in Drug Substances,” 64 Fed. Reg. 67917 (1999). Unless an impurity has been tested for safety, is in a composition proven to be safe in clinical trials, or is a human metabolite, the CDER further recommends that the drug applicant reduce the amount of the impurity in the active ingredient to below 0.1%. Therefore, in order to study the pharmacology and toxicology of such impurities, there is a need to isolate impurities in drug substances.
In order to obtain marketing approval for a new drug product, manufacturers must submit to the regulatory authority evidence that the product is acceptable for administration to humans. Such a submission must include, among other things, analytical data showing the impurity profile of the product to demonstrate that the impurities are either absent, or present in a negligible amount. Therefore, there is a need for analytical methods to detect impurities, and for reference standards to identify and assay those impurities.
Generally, impurities (side products, byproducts, and adjunct reagents) are identified spectroscopically and by other physical methods and then the impurities are associated with a peak position in a chromatogram (or a spot on a TLC plate). (Strobel p. 953) (Strobel, H. A.; Heineman, W. R., Chemical Instrumentation: A Systematic Approach, 3rd dd. (Wiley & Sons: New York 1989)). Thereafter, the impurity can be identified by its position in the chromatogram, which is conventionally measured in minutes between injection of the sample on the column and elution of the particular component through the detector, known as the “retention time.” This time period varies daily based upon the condition of the instrumentation and many other factors. To mitigate the effect that such variations have upon accurate identification of an impurity, practitioners use “relative retention time” (“RRT”) to identify impurities. (Strobel p. 922). The RRT of an impurity is its retention time divided by the retention time of some reference marker. In theory, mycophenolate mofetil itself could be used as the reference marker, but as a practical matter it is present in such overwhelming proportion in the mixture that it tends to saturate the column, leading to irreproducible retention times, i.e., the maximum of the peak corresponding to mycophenolate mofetil tends to wander (Strobel FIG. 24.8(b) p. 879, contains an illustration of the sort of asymmetric peak that is observed when a column is overloaded). Thus, it is sometimes desirable to select an alternative compound that is added to, or is present in, the mixture in an amount significant enough to be detectable and sufficiently low as not to saturate the column and to use that compound as the reference marker.
Researchers and developers in drug manufacturing understand that a compound in a relatively pure state can be used as a “reference standard” (a “reference marker” is similar to a reference standard but it is used for qualitative analysis) to quantify the amount of the compound in an unknown mixture. When the compound is used as an “external standard,” a solution of a known concentration of the compound is analyzed by the same technique as the unknown mixture. (Strobel p. 924, Snyder p. 549) (Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed. (John Wiley & Sons: New York 1979)). The amount of the compound in the mixture can be determined by comparing the magnitude of the detector response. See also U.S. Pat. No. 6,333,198, incorporated herein by reference.
The reference standard compound also can be used to quantify the amount of another compound in the mixture if the “response factor,” which compensates for differences in the sensitivity of the detector to the two compounds, has been predetermined. (Strobel p. 894). For this purpose, the reference standard compound may be added directly to the mixture, in which case it is called an “internal standard.” (Strobel p. 925, Snyder p. 552).
The reference standard compound can even be used as an internal standard when the unknown mixture contains some of the reference standard compound by using a technique called “standard addition,” wherein at least two samples are prepared by adding known and differing amounts of the internal standard. (Strobel pp. 391-393, Snyder pp. 571, 572). The proportion of detector response due to the reference standard compound that is originally in the mixture can be determined by extrapolation of a plot of detector response versus the amount of the reference standard compound that was added to each of the samples to zero. (e.g. Strobel, FIG. 11.4 p. 392).
Esterification of MPA is known. (e.g. in Synthetic Organic Chemistry by R. B. Wagner and H. D. Zook, Wiley, New York, 1956, see pages 479-532). U.S. Pat. No. 4,753,935 first disclosed mycophenolate mofetil. However, the synthetic process to prepare the ester results in various impurities.
PHARMAEUROPA vol 15 No 4 October 2003 published a list of possible impurities of Mycophenolate Mofetil (from A to H). The present invention relates to a new impurity whose presence was observed in Mycophenolate Mofetil and which is not included in this list. This impurity is useful as a reference standard in preparation of highly pure mycophenolate mofetil.